1. Field of the Invention
The present invention provides a method for preparing a peptide. In particular, the present invention provides a method of making an intermediate for preparation of Luteinizing Hormone-Releasing Hormone (hereinafter also referred to as “LHRH”) antagonists.
2. Discussion of the Background
Heretofore, several LHRH antagonists have been identified, which include:
Cetrolelix:
Ac-D-2Nal-D-4ClPhe-D-3Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2;
Antarelix:
Ac-D-2 Nal-D-4ClPhe-D-3Pal-Ser-Tyr-Hci-Leu-Lys(iPr)-Pro-D-Ala-NH2;
Abarelix:
Ac-D-2Nal-D-4ClPhe-D-3Pal-Ser-Tyr(NCH3)-D-Asn-Leu-Lys(iPr)-Pro-D-Ala-NH2;
Ganirelix:
Ac-D-2Nal-D-4ClPhe-D-3Pal-Ser-Tyr-D-Lys(C(NHEt)2)-Leu-Lys(C(NHEt)2)-Pro-D-Ala-NH2.
An important intermediate for the aforementioned LHRH antagonists is a tripeptide represented by the formula [I]:Ac-D-2Nal-D-4ClPhe-D-3Pal-OH  [I]
An example of such a tripeptide is N-acetyl-D-2-naphthylalanyl-D-4-chlorophenylalanyl-D-3-pyridylalanine.
In formula [I], and in the present specification,
“Ac” represents acetyl,
“2Nal” represents a divalent group represented by the formula:

“4ClPhe” represents a divalent group represented by the formula:

“3Pal” represents a divalent group represented by the formula:

“D” is a prefix for the D-configuration, and
“L” is a prefix for the L-configuration.
In the method of preparing the above-described tripeptide represented by the formula [I], Boc-D-4ClPhe-OH [in the present specification, “Boc” represents a tert-butoxycarbonyl group], for example, is activated by a reaction with N-hydroxysuccinimide, and then condensed with H-D-3Pal-OH 2HCl to yield a dipeptide Boc-D-4ClPhe-D-3Pal-OH. Subsequently, by Boc-deprotection, H-D-4ClPhe-D-3Pal-OH is prepared and is then condensed with Boc-D-2Nal-OH, which was previously separately activated (for example, by a reaction with N-hydroxysuccinimide), to yield Boc-D-2Nal-D-4ClPhe-D-3Pal-OH. Boc-deprotection is performed again and the N-terminal is acetylated to yield the desired tripeptide (see WO 03/055902).
However, because this method employs Boc-D-4ClPhe-OH and Boc-D-2Nal-OH, the Boc group must be deprotected and/or converted to an acetyl group, resulting in an increased number of steps and other problems. Additionally, the Boc-deprotected N-unprotected peptide is highly hygroscopic and hence difficult to handle. Therefore, the purity of the resulting tripeptide is as low as 93.9% (HPLC Area %).
In an alternative synthesis method, Boc-D-4ClPhe-OH and H-D-3Pal-OMe 2HCl are condensed to prepare a dipeptide Boc-D-4ClPhe-D-3Pal-OMe, which is Boc-deprotected and then condensed with Boc-D-2Nal-OH to yield the tripeptide Boc-D-2Nal-D-4ClPhe-D-3Pal-OMe (85.4% yield) (see WO 97/034924). However, to obtain the tripeptide represented by the formula [I], the Boc group must be converted to an acetyl group as described above, and the process unavoidably involves a highly hygroscopic N-unprotected peptide.
Still another method is available wherein condensation of Boc-D-2Nal-OH and H-D-4ClPhe-OMe HCl is followed by Boc-deprotection and then acetylation to provide a dipeptide Ac-D-2Nal-D-4ClPhe-OMe (85.7% yield), which dipeptide is used (see WO 99/026964). Again, this method requires the conversion of the protecting group (Boc→Ac), resulting in an increased number of steps. Although it is possible to hydrolyze this dipeptide to yield Ac-D-2Nal-D-4ClPhe-OH (88.0% yield), which can be used to synthesize the tripeptide represented by the formula [I], the D-4ClPhe moiety racemizes, resulting in the contamination of 2.6% of Ac-D-2Nal-L-4ClPhe-OH and an as low purity as 93.7% (HPLC Area %) (see U.S. Pat. No. 6,492,490).
It has generally been reported that racemization often proceeds when using an acetylated amino acid as a starting material (see Int. J. Peptide Protein Res. 28, 1986, 444-449; Helvetica Chimica Acta, 74, 1991, 617-627). As racemization proceeds, a complex extraction step and/or column purification step to remove the diastereomer would be required, resulting in an increased number of steps.
Accordingly, there remains a critical demand for a method of preparing intermediates for LHRH antagonists (i.e., the tripeptide compound represented by the formula [I]) in which the process comprises fewer steps, as well as a method that enables the preparation of the desired intermediate in high yield and high purity.